1. Field of the Invention
The present invention relates to plant genetic engineering. More particularly, to a method for constructing an artificial polynucleotide and methods of use to reduce transgene silencing in plants. The invention also relates to the plant cells containing the artificial polynucleotide in which a plant cell is transformed to express the artificial polynucleotide and the plant regenerated therefrom.
2. Description of the Related Art
Heterologous genes may be isolated from a source other than the plant into which it will is be transformed or they may be modified or designed to have different or improved qualities. Particularly desirable traits or qualities of interest for plant genetic engineering would include but are not limited to resistance to insects, fungal diseases, and other pests and disease-causing agents, tolerances to herbicides, enhanced stability or shelf-life, yield, environmental stress tolerances, and nutritional enhancements.
Traditional molecular biological methods for generating novel genes and proteins generally involved random or directed mutagenesis. An example of random mutagenesis is a recombination technique known as “DNA shuffling” as disclosed in U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,837,458 and International Applications WO 98/31837, WO 99/65927, the entirety of all of which is incorporated herein by reference. An alternative method of molecular evolution involves a staggered extension process (StEP) for in vitro mutagenesis and recombination of nucleic acid molecule sequences, as disclosed in U.S. Pat. No. 5,965,408, incorporated herein by reference. An example of directed mutagenesis is the introduction of a point mutation at a specific site in a polypeptide.
An alternative approach, useful when the heterologous gene is from a non-plant source, is to design an artificial insecticidal gene that uses the most often used codon in maize plant codon usage table (Koziel et al., 1993, Biotechnology 11, 194-200). Fischhoff and Perlak (U.S. Pat. No. 5,500,365, incorporated herein by reference) report higher expression of Bacillus thuringiensis (Bt) insecticidal protein compared in crop plants when the polynucleotide sequence was modified to reduce the occurrence of destabilizing sequences. It was necessary to modify the wild type Bt polynucleotide sequence because the wild type full length Bt polynucleotide did not express sufficient levels of insecticidal protein in plants to be agronomically useful.
Heterologous genes are cloned into vectors suitable for plant transformation. Transformation and regeneration techniques useful to incorporate heterologous genes into a plant's genome are well known in the art. The gene can then be expressed in the plant cell to exhibit the added characteristic or trait. However, heterologous genes that normally express well as transgenes may experience gene silencing when more than one copy of the same genes are expressed in the same plant. This may occur when a first heterologous gene is too similar to an endogenous gene DNA sequence in the plant. Other examples include when a transgenic plant is subsequently crossed to other transgenic plants having the same or similar transgenes or when the transgenic plant is retransformed with a plant expression cassette that contains the same or is similar gene. Similarly, gene silencing may occur if trait stacking employs the same genetic elements used to direct expression of the transgene gene of interest. In order to stack traits, stable transgenic lines should be done with different combinations of genes and genetic elements to avoid gene silencing.
N-phosphonomethylglycine, also known as glyphosate, is a well-known herbicide that has activity on a broad spectrum of plant species. Glyphosate is the active ingredient of Roundup® (Monsanto Co.), a safe herbicide having a desirably short half-life in the environment. When applied to a plant surface, glyphosate moves systemically through the plant. Glyphosate is phytotoxic due to its inhibition of the shikimic acid pathway, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS).
Glyphosate tolerance can also be achieved by the expression of EPSPS variants that have lower affinity for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (U.S. Pat. No. 5,633,435, herein incorporated by reference). Enzymes that degrade glyphosate in plant tissues (U.S. Pat. No. 5,463,175) are also capable of conferring cellular tolerance to glyphosate. Such genes are used for the production of transgenic crops that are tolerant to glyphosate, thereby allowing glyphosate to be used for effective weed control with minimal concern of crop damage. For example, glyphosate tolerance has been genetically engineered into corn (U.S. Pat. No. 5,554,798), wheat (U.S. Patent Application No. 20020062503), soybean (U.S. Patent Application No. 20020157139) and canola (WO 9204449), all of which are incorporated by reference. The transgenes for glyphosate tolerance and the transgenes for tolerance to other herbicides, e.g. bar gene, (Toki et al. Plant Physiol., 100:1503-1507, 1992; Thompson et al. EMBO J. 6:2519-2523, 1987, phosphinothricin acetyltransferase, BAR gene isolated from Streptomyces; DeBlock et al. EMBO J., 6:2513-2522, 1987, glufosinate herbicide) are also useful as selectable markers or scorable markers and can provide a useful phenotype for selection of plants linked with other agronomically useful traits.
What is needed in the art are methods to design genes for expression in plants to improve agronomically useful traits that avoid gene silencing when multiple copies are inserted and recombination with endogenous plant genes.
BRIEF DESCRIPTION OF SEQUENCE LISTINGSEQ ID NO: 1OsEPSPS_TIPSA rice EPSPS protein sequence modified tobe glyphosate resistant, with chloroplasttransit peptide.SEQ ID NO: 2OsEPSPS_NatPolynucleotide sequence of a rice nativeEPSPS polynucleotide modified to encode aglyphosate resistant protein.SEQ ID NO: 3OsEPSPS_ATPolynucleotide sequence of an artificial riceEPSPS polynucleotide using the Arabidopsiscodon usage table and the methods of thepresent invention, and further modified toencode a glyphosate resistant protein.SEQ ID NO: 4OsEPSPS_ZMPolynucleotide sequence of an artificial riceEPSPS polynucleotide using the Zea mayscodon usage table and the methods of thepresent invention, and further modified toencode a glyphosate resistant protein.SEQ ID NO: 5GmEPSPS_IKSA soybean EPSPS protein sequence modifiedto be glyphosate resistant, with chloroplasttransit peptide.SEQ ID NO: 6GmEPSPS_NatPolynucleotide sequence of a soybean nativeEPSPS polynucleotide modified to encode aglyphosate resistant protein.SEQ ID NO: 7GmEPSPS_GMPolynucleotide sequence of an artificialsoybean EPSPS polynucleotide using theGlycine max codon usage table and themethods of the present invention, and furthermodified to encode a glyphosate resistantprotein.SEQ ID NO: 8ZmEPSPS_TIPSA corn EPSPS protein sequence modified tobe glyphosate resistant, with chloroplasttransit peptide.SEQ ID NO: 9ZmEPSPS_NatPolynucleotide sequence of a corn nativeEPSPS polynucleotide modified to encode aglyphosate resistant protein.SEQ ID NO: 10ZmEPSPS_ZMPolynucleotide sequence of an artificial cornEPSPS polynucleotide using the Zea mayscodon usage table and the methods of thepresent invention, and further modified toencode a glyphosate resistant protein.SEQ ID NO: 11CTP2Protein sequence of the chloroplast transitpeptide 2 from Arabidopsis EPSPS gene.SEQ ID NO: 12CTP2_NatPolynucleotide sequence of the chloroplasttransit peptide from Arabidopsis EPSPS.SEQ ID NO: 13CTP2_ATPolynucleotide sequence of an artificialpolynucleotide encoding the CTP2 using theArabidopsis codon usage table and themethods of the present invention.SEQ ID NO: 14CTP2_ZMPolynucleotide sequence of an artificialpolynucleotide encoding the CTP2 using theZea mays codon usage table and the methodsof the present invention.SEQ ID NO: 15CP4EPSPSThe protein sequence of the glyphosateresistant EPSPS protein from Agrobacteriumstrain CP4.SEQ ID NO: 16CP4EPSPS_NatPolynucleotide sequence of the nativepolynucleotide encoding the CP4EPSPSprotein (U.S. Pat. No. 5,633,435).SEQ ID NO: 17CP4EPSPS_ATPolynucleotide sequence of an artificialpolynucleotide encoding the CP4EPSPSprotein using the Arabidopsis codon usagetable and the methods of the presentinvention.SEQ ID NO: 18CP4EPSPS_ZMPolynucleotide sequence of an artificialpolynucleotide encoding the CP4EPSPSprotein using the Zea mays codon usage tableand the methods of the present invention.SEQ ID NO: 19BAR1The protein sequence of a phosphinothricinacetyltransferase.SEQ ID NO: 20BAR1_NatPolynucleotide sequence of the nativepolynucleotide isolated from Streptomycesencoding the phosphinothricinacetyltransferase.SEQ ID NO: 21BAR1_ATPolynucleotide sequence of an artificialpolynucleotide encoding the phosphinothricinacetyltransferase using the Arabidopsis codonusage table and the methods of the presentinvention.SEQ ID NO: 22BAR1_ZMPolynucleotide sequence of an artificialpolynucleotide encoding the phosphinothricinacetyltransferase using the Zea mays codonusage table and the methods of the presentinvention.SEQ ID NO: 23CP4EPSPS_SynPolynucleotide sequence of an artificialpolynucleotide with dicot codon bias.SEQ ID NO: 24CP4EPSPS_AT_p1DNA primer molecule diagnostic for theCP4EPSPS_AT polynucleotide.SEQ ID NO: 25CP4EPSPS_AT_p2DNA primer molecule diagnostic for theCP4EPSPS_AT polynucleotide.SEQ ID NO: 26CP4EPSPS_ZM_p1DNA primer molecule diagnostic for theCP4EPSPS_ZM polynucleotide.SEQ ID NO: 27CP4EPSPS_ZM_p2DNA primer molecule diagnostic for theCP4EPSPS_ZM polynucleotide.SEQ ID NO: 28CP4EPSPS_Nat_p1DNA primer molecule diagnostic for theCP4EPSPS_Nat polynucleotide.SEQ ID NO: 29CP4EPSPS_Nat_p2DNA primer molecule diagnostic for theCP4EPSPS_Nat polynucleotide.SEQ ID NO: 30CP4EPSPS_Syn_p1DNA primer molecule diagnostic for theCP4EPSPS_Syn polynucleotide.SEQ ID NO: 31CP4EPSPS_Syn_p2DNA primer molecule diagnostic for theCP4EPSPS_Syn polynucleotide.SEQ ID NO: 32ZmAdh1 primer1Control primer 1 diagnostic for endogenouscorn Adh1 gene.SEQ ID NO: 33ZmAdh1 primer2Control primer 2 diagnostic for endogenouscorn Adh1 gene.SEQ ID NO: 34GNAGIAMKSMotif providing glyphosate resistance to aplant EPSPS.SEQ ID NO: 35CTPEPSPSCP4_GMPolynucleotide sequence of an artificialpolynucleotide encoding the CP4EPSPSprotein using the Glycine max codon usagetable.